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J. Biol. Chem., Vol. 275, Issue 34, 26376-26384, August 25, 2000

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The Protein-tyrosine Phosphatase SHP-1 Binds to and Dephosphorylates p120 Catenin^*

Heike Keilhack, Ulf Hellman^§, Jolanda van Hengel^¶, Frans van Roy^¶**, Jasminka Godovac-Zimmermann^§§, and Frank-D. Böhmer^¶¶

From the  Research Unit "Molecular Cell Biology," Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Strasse 1, D-07747 Jena, Germany, the ^§ Ludwig Institute for Cancer Research, Uppsala Branch, SE-75124 Uppsala, Sweden, the ^¶ Molecular Cell Biology Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Belgium, and the  Institute for Molecular Biotechnology, Beutenbergstrasse 11, D-07745 Jena, Germany

Received for publication, February 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A prominent tyrosine-phosphorylated protein of ~100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120^ctn). Different p120^ctn isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120^ctn tyrosine phosphorylation. Despite strong phosphorylation, p120^ctn isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120^ctn in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120^ctn exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120^ctn, which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120^ctn interaction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The protein-tyrosine phosphatase (PTP)¹ SHP-1 (hematopoietic cell phosphatase, SH-PTP1, and PTP1C) (1) is expressed in hematopoietic and epithelial cells (for reviews, see Refs. 2-5). The epithelial and hematopoietic variants of SHP-1 differ in the sequence of 4 amino acids at the amino terminus (6). SHP-1 contains two SH2 domains that target the enzyme to cellular substrates. Binding of SHP-1 to tyrosine-phosphorylated proteins results in activation of SHP-1. In consequence, the binding partner may become dephosphorylated, including the phosphotyrosine residues required for binding, leading eventually to decomposition of the complex. Alternatively and in addition, SHP-1 can dephosphorylate phosphoproteins in the vicinity of the SH2 domain-binding partner, e.g. signaling molecules acting downstream of a receptor that binds SHP-1 or vice versa. As revealed by analysis of chimeras of SHP-1 and the structurally closely related PTP SHP-2, selectivity of cellular actions of SHP-1 is also determined by the catalytic domain specificity, in addition to the SH2 domain specificity (7, 8). In hematopoietic cells, negative modulation of signal transduction of various cytokine receptors by SHP-1 has been firmly established, including signaling of the erythropoietin receptor (9), the colony-stimulating factor-1 receptor (10), and the Kit/stem cell factor receptor (11, 12). SHP-1 mediates signals of inhibitory immunoreceptors (13-19); and in macrophages, SHP-1 has been shown to negatively regulate integrin-mediated cell adhesion (20). A family of tyrosine-phosphorylated transmembrane proteins, designated SIRP or SHP substrate (SHPS), has been described to bind SHP-1 and SHP-2 and is believed to regulate the cellular actions of both PTPs (21-23). Little is known about the biological role of SHP-1 in epithelial cells, although the existence of an epithelium-specific isoform of SHP-1 (6) is suggestive of specific functions in these cells.

Cell-cell adhesion of epithelial cells involves homophilic interactions of cadherins (24). Cadherin-mediated cell-cell adhesion is important for development and maintenance of epithelial tissue integrity (25), and its disturbance contributes to the invasive and metastatic phenotype of epithelial tumors (26, 27). Through their intracellular domains, cadherins associate with molecules of the Armadillo superfamily, including -catenin and p120 catenin (p120^ctn). The former binds to the carboxyl-terminal domain of E-cadherin and links this to the vinculin-related actin-binding -catenin (28, 29). In contrast, p120^ctn binds to the juxtamembrane domain of the cytoplasmic part of E-cadherin (30-32). Cadherin-catenin association is essential for the functionality of cell-cell adhesion complexes (33). -Catenin mediates association with the actin cytoskeleton via the -catenin bridge (34), but also participates in intracellular signaling (35). Thus, -catenin can translocate to the nucleus to cooperate with transcription factors of the LEF/TCF family (36). The stability of cadherin-catenin complexes and thereby the function of cell-cell adhesion complexes and possibly the signaling function of catenins appear to be regulated by tyrosine phosphorylation (37). -Catenin can be phosphorylated, for example, upon Src activation (38) or epidermal growth factor receptor (EGFR) activation (39), leading to dissociation of the cadherin-catenin complex. Elevated -catenin phosphorylation has been observed in epithelial tumors (40). A role of tyrosine phosphorylation in the regulation of catenin function and cadherin-mediated cell-cell adhesion can also be inferred from the presence of PTP activity in cadherin-catenin complexes and from the observation that the transmembrane PTPs PTPµ, PTP, and LAR localize to cell-cell adhesion complexes and can associate with -catenin (41-43). Pronounced tyrosine phosphorylation has also been observed for p120^ctn (44, 45). In addition to Src, the cytoplasmic tyrosine kinase FER has been shown to phosphorylate p120^ctn (46, 47). The precise function of p120^ctn is, however, still not known. It has been reported to be involved in positive regulation of cadherin clustering and cell-cell adhesion, but also in cell motility (48, 49). Recent publications reveal apparently conflicting roles for p120^ctn in cadherin-mediated adhesion. Phosphorylated p120^ctn isoforms have been proposed to negatively modulate cadherin function and cell-cell adhesion in human colon carcinoma cells (50) and transfected mouse L-cells (51). On the other hand, Yap et al. (48) and Thoreson et al. (32) showed that the juxtamembrane domain of cadherins supports clustering and promotes strong cell-cell adhesion. p120^ctn isoforms lacking sequences encoded by exon 20, designated exon B, have the capacity to localize to the nucleus, suggesting nuclear functions of p120^ctn (52). Indeed, p120^ctn was shown to form a complex with a zinc-finger transcription factor named Kaiso (53). The physiological role for tyrosine phosphorylation of p120^ctn is currently unknown, but is likely to regulate p120^ctn function. Inducible FER overexpression in Rat-2 fibroblasts led to reduced association of cadherin with -and -catenins, suggesting that FER-mediated tyrosine phosphorylation of p120^ctn may negatively regulate the stability of cell-cell adhesion complexes (46).

We show here that p120^ctn is one of the main binding partners for the PTP SHP-1 in EGF-stimulated A431 cells and a substrate for SHP-1 in these cells. Different isoforms of p120^ctn exhibit differential SHP-1 interaction potential, and this appears to be partly related to their capacity to become tyrosine-phosphorylated in an EGF-dependent manner. We propose that SHP-1 may regulate the function of p120^ctn by its dephosphorylation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and Reagents-- EGF was purchased from Pepro Tech, Inc., (Rocky Hill, NJ). Polyclonal anti-phosphotyrosine antibodies and monoclonal anti-p120^ctn antibodies were obtained from Transduction Laboratories (Lexington, KY). Monoclonal anti-phosphotyrosine antibodies covalently coupled to Sepharose (clone PT-66) were from Sigma. Polyclonal anti-SHP-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Vav2 antibodies were kindly provided by Dr. X. Bustelo (State University of New York, Stony Brook, NY).

Cells, DNAs, and Protein Expression-- A431 cells were from American Type Culture Collection (CRL 1555; Manassas, VA), and human embryonal kidney 293 cells were kindly provided by Dr. A. Ullrich (Max-Planck Institute for Biochemistry, Martinsried, Germany). Both cell lines were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% fetal calf serum. A cDNA for human SHP-1 (epithelial form) was generously provided by Drs. A. Ullrich and R. Lammers (Max-Planck Institute for Biochemistry). Generation of A431 cell lines with inducible SHP-1 expression is described elsewhere (54). Expression constructs for the various p120^ctn isoforms (52) and constructs for GST fusion proteins of SHP-1 and SHP-1 mutants (55) were described earlier. GST fusion protein purification (55) and transient transfections (56) were performed as described.

GST Pull-down Assays and Deglycosylation Assay-- A431 cells were grown in 94-mm dishes to ~70% confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cells were starved for 16 h using serum-free Dulbecco's modified Eagle's medium and subsequently stimulated with 100 ng/ml EGF for 5 min and lysed in 700 µl of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and 0.5 mM sodium pervanadate). Note that this Triton X-100 concentration appeared to be critical to observe the SHP-1-p120^ctn association, as little if any association could be seen at 1% Triton X-100. The lysates were centrifuged at 25,000 × g for 20 min. To investigate binding of tyrosine-phosphorylated proteins to SHP-1, 50 µg of GST-SHP-1 WT, GST-SHP-1 R32K, GST-SHP-1 R138K, GST-SHP-1 R32K,R138K, or GST proteins were coupled to 30 µl of glutathione-Sepharose (Amersham Pharmacia Biotech). The A431 cell lysates were incubated for 2 h at 4 °C with the immobilized fusion proteins by end-over-end rotation. The beads were washed three times with HNGT buffer (20 mM Hepes (pH 7.4), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, and 0.5 mM sodium pervanadate). Then, 60 µl of 2× Laemmli buffer were added, and the mixture was boiled for 5 min. Bound proteins were visualized by SDS-PAGE (7.5% acrylamide gels) and immunoblotting as described previously (7, 55) using anti-phosphotyrosine antibodies.

To identify glycosylated SHP-1-binding tyrosine-phosphorylated proteins, a deglycosylation assay was used. Thirty µl of glutathione beads with associated proteins (as described above) were incubated with 45 µl of solution containing 0.1% SDS and 1% -mercaptoethanol for 5 min at 95 °C. Then, 45 µl of 2× reaction buffer (100 mM sodium phosphate (pH 7), 40 mM EDTA, 1% -mercaptoethanol, and 2% Triton X-100) and 0.5 units of endoglycosidases F/N (Roche Molecular Biochemicals) were added. The reaction was incubated for 16 h at 37 °C. Subsequently, 18 µl of 6× Laemmli buffer were added, and the mixture was boiled for 5 min. Proteins were analyzed by SDS-PAGE (7.5% acrylamide gels) and immunoblotting using anti-phosphotyrosine antibodies.

To investigate whether the interaction of SHP-1 and p120^ctn is direct or not, a sequential immunoprecipitation approach was used. This was done basically as described previously (55). Briefly, subconfluent A431 cells (94-mm dishes) were stimulated with EGF (100 ng/ml) for 5 min and lysed in 700 µl of lysis buffer. p120^ctn was immunoprecipitated in its tyrosine-phosphorylated form using anti-phosphotyrosine antibodies covalently coupled to Sepharose. The bound proteins were denatured by boiling in 50 mM Hepes (pH 7.5), 1% SDS, and 1% -mercaptoethanol for 5 min and subsequently partially renatured by diluting the sample 1:20 with lysis buffer and incubating at 4 °C for 30 min. Thereafter, the partially renatured lysates were used in GST pull-down assays employing 50 µg of GST-SHP-1 WT, GST-SH2 (isolated tandem SH2 domains of SHP-1 as a GST fusion protein), GST-SHP-1 CS (catalytically inactive C455S mutant of SHP-1), or GST (as a control). The associated proteins were visualized by SDS-PAGE (7.5% acrylamide gels) and immunoblotting.

To monitor binding of the p120^ctn isoforms to SHP-1, cDNAs for isoforms 1A, 2A, 3A, 4A, 3N, 3AB, 3AC, 3ABC, 3B, 3C, and 3BC (cloned into expression vector pEFBOS) (52, 57) were cotransfected with pRK5RS-EGFR (56, 58) into 293 cells (94-mm dishes). The cells were stimulated with EGF (100 ng/ml for 5 min) and lysed in 700 µl of lysis buffer. The lysates were centrifuged at 25,000 × g for 20 min and subsequently incubated with end-over-end rotation at 4 °C with 10 µg of GST-SHP-1 immobilized on 30 µl of glutathione-Sepharose for 2 h. The beads were washed with HNGT buffer, and noncovalently bound proteins were extracted by boiling for 5 min in the presence of 60 µl of 2× Laemmli buffer. The proteins were visualized by SDS-PAGE (7.5% acrylamide gels) and immunoblotting.

Partial Purification of p120^ctn Isoform 3A (pp100)-- A431 cells were cultured at large scale in 94-mm dishes. The cells were stimulated with EGF (100 ng/ml) for 5 min when they reached 70% confluence. The cells were lysed in 700 µl of lysis buffer/plate. The lysates were centrifuged at 25,000 × g for 20 min, pooled, and loaded onto a 0.5-ml anti-phosphotyrosine-Sepharose column equilibrated in buffer R1 (50 mM Tris-HCl (pH 8), 0.1% Triton X-100, 1 mM Na₃VO₄, and 0.5 M NaCl). The column was washed with 10 ml of buffer R1 and subsequently with 10 ml of buffer R2 (50 mM Tris-HCl (pH 8), 0.1% Triton X-100, and 1 mM Na₃VO₄). The bound phosphoproteins were eluted with 3 ml of 20 mM phenyl phosphate dissolved in buffer R2. Thereafter, the eluate was applied to a MonoQ HR5/5 column (Amersham Pharmacia Biotech) equilibrated in buffer R2 and connected to a fast protein liquid chromatography system. The bound proteins were eluted with a linear NaCl gradient (0-500 mM NaCl within 20 ml). The fractions were analyzed by SDS-PAGE and silver staining, and positive fractions were identified by running GST pull-down assays using GST-SHP-1 as described above. These positive fractions were pooled; 80% acetone was added; and the mixture was kept on ice for 1 h. The precipitated proteins were pelleted by centrifugation at 30,000 × g for 30 min at 4 °C. The pellet was dried and dissolved in 2× Laemmli buffer. The proteins were resolved by SDS-PAGE (7.5% acrylamide gels). Gels were stained with Coomassie Blue, and the respective bands were excised and used for structure analysis.

In-gel Digestion, Mass Spectrometric Analysis, and Edman Sequencing-- The excised band was prepared for and subjected to in-gel digestion as described (59). In brief, after washing with ammonium bicarbonate and acetonitrile, the gel piece was completely dried, and a solution containing modified porcine trypsin (sequence-grade; Promega, Madison, WI) was allowed to soak into the gel piece. After overnight incubation at 30 °C, generated peptides were recovered by extraction. The peptide mixture was analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using a Bruker Biflex III instrument equipped with delayed extraction and reflector. The sample was prepared by the dried droplet technique using -cyano-4-hydroxycinnamic acid as the matrix. The instrument was externally calibrated using angiotensin II (MH⁺ 1046.54) and adrenocorticotropic hormone fragment 18-39 (MH⁺ 2465.20). The peptide mass fingerprinting analysis was done using ProFound Version 4.7.5.

For Edman degradation, peptides were isolated by microbore reversed-phase liquid chromatography on a 1 × 150-mm Kromasil C18 column operated in the SMART system (Amersham Pharmacia Biotech, Uppsala, Sweden). Selected fractions were subjected to amino acid sequence analysis in a Procise 494 instrument (PE-Biosystems, Foster City, CA) following the manufacturer's instructions.

Expression Analysis of p120^ctn Isoforms by RT-PCR-- RNA was isolated from A431 cells using the RNA-easy kit (QIAGEN Inc., Hilden, Germany) according to the manufacturer's instructions. For RT-PCR, cDNA was synthesized as described previously (57). The PCR mixtures contained template cDNA, 25 pmol of p120^ctn-specific primers, 200 mM dXTPs, 2.5 mM MgCl₂, and the buffer supplied with the Taq DNA polymerase (Life Technologies, Inc., Gent, Belgium). Taq DNA polymerase was used at 0.5 units/reaction. The following primers were used: primer set EX1F6 plus EX5R1 covers the 5'-end (alternative start codons) of the p120^ctn cDNAs; set EX10F2 plus EX12R1 generates a product of 175 base pairs when exon C is absent and one of 193 base pairs when exon C is present; set EX15F1 and EX20R1 amplifies only cDNA fragments that contain exon B; and set EX18F1 and EX21R3 amplifies only fragments that contain exon A (a 557-base pair product in the presence of exon B and a 470-base pair product when exon B sequences are spliced out). The primer sequences and the corresponding cycling conditions were described before (57).

Immunoprecipitation and Dephosphorylation Assays-- To monitor the interaction of p120^ctn and SHP-1 in intact cells, A431 cells with inducible SHP-1 expression were used (54). The cells were grown in the absence of anhydrotetracycline (ATc) in 94-mm dishes to 70% confluence and then either stimulated with EGF (100 ng/ml) for different time points or left unstimulated and lysed in 700 µl of lysis buffer Z (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 10 mM NaF, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, and 1 mM zinc acetate). The lysates were cleared by centrifugation at 25,000 × g for 20 min at 4 °C and subjected to immunoprecipitation using monoclonal anti-SHP-1 antibodies (1 µg/reaction). The immunoprecipitations were incubated with end-over-end rotation for 2 h at 4 °C. Subsequently, 15 µl of protein A-Sepharose (Amersham Pharmacia Biotech) were added, and the mixtures were incubated for another 30 min with end-over-end rotation at 4 °C. The beads were washed three times with HNGT buffer containing 1 mM zinc acetate in place of the sodium pervanadate, and the noncovalently bound proteins were extracted by boiling in 40 µl of 2× Laemmli buffer. The proteins were resolved by SDS-PAGE (7.5% acrylamide gels) and visualized by immunoblotting.

To analyze the dependence of tyrosine phosphorylation of p120^ctn on the absence or presence of SHP-1, SHP-1-expressing A431 cell lines and mock-transfected A431 control cell lines were cultured for 4 days in the presence or absence of 100 ng/ml ATc (six-well plates) until the cells reached ~70% confluence. The cells were stimulated for different time points with EGF (100 ng/ml) and lysed in 200 µl of lysis buffer/well. The lysates were centrifuged at 25,000 × g for 20 min, and p120^ctn was immunoprecipitated using anti-p120^ctn antibodies. The p120^ctn tyrosine phosphorylation was monitored after SDS-PAGE by immunoblotting using anti-phosphotyrosine antibodies.

To visualize tyrosine phosphorylation efficiency of the different p120^ctn isoforms by EGFR, cDNAs of these isoforms were cotransfected with EGFR cDNA into 293 cells in six-well plates (0.5 µg of pRK5-EGFR and 3.5 µg of pEFBOS-p120^ctn). The cells were stimulated with 100 ng/ml EGF for 5 min and lysed in 200 µl of lysis buffer/well. After centrifugation at 25,000 × g for 20 min at 4 °C, lysate aliquots were loaded onto SDS-polyacrylamide gels, and the tyrosine phosphorylation of the different p120^ctn isoforms was monitored by immunoblotting.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Interaction of SHP-1 with a 100-kDa Phosphoprotein in EGF-stimulated Cells-- To identify new interaction partners for SHP-1 in epithelial cells, we performed GST pull-down experiments using GST-SHP-1 and lysates of EGF-stimulated A431 cells. We observed precipitation of autophosphorylated EGFR, as described earlier, and of a prominent tyrosine-phosphorylated protein of ~100 kDa (Fig. 1A), designated operationally as pp100. This protein was apparently one of the major tyrosine-phosphorylated proteins after EGF stimulation in A431 cells. We tested whether the precipitation of pp100 would exhibit any specificity with respect to the two SH2 domains of SHP-1 employing GST-SHP-1 fusions with inactivated SH2 domains (Fig. 1B, left panel). Inactivation of both SH2 domains by point mutation abolished the association of pp100 as well as of EGFR with the GST-SHP-1 fusion protein. Point mutation of the N-terminal SH2 domain completely abrogated interaction with pp100, whereas EGFR was still precipitated to some extent. Point mutation of the C-terminal SH2 domain reduced the association of pp100, however, to a lesser extent than the association of EGFR. Taken together, these experiments indicate that binding of pp100 to SHP-1 is mediated mainly by the N-terminal SH2 domain. The coprecipitation of EGFR with GST-SHP-1 mutants exhibits a different pattern, suggesting that the association of pp100 with SHP-1 is independent of the association of EGFR with SHP-1. Further evidence for a lack of association between pp100 and EGFR comes from their independent behavior upon wheat germ agglutinin affinity chromatography, with EGFR largely recovered in the wheat germ agglutinin-bound fraction and pp100 appearing in the flow-through fraction (data not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Binding of a 100-kDa tyrosine-phosphorylated protein from EGF-stimulated A431 cells to SHP-1 and its identification as p120ctn. Subconfluent A431 cells were stimulated for 5 min with EGF and then lysed. A: the lysates were incubated with immobilized GST-SHP-1 or immobilized GST, and the bound tyrosine-phosphorylated proteins were visualized by immunoblotting using anti-phosphotyrosine (anti-PY) antibodies. B: left panel, GST alone (negative control) or a fusion protein of GST and wild-type SHP-1 (GST-SHP-1), SHP-1 with an inactivated N-terminal SH2 domain (GST-SHP-1 R32K), SHP-1 with an inactivated C-terminal SH2 domain (GST-SHP-1 R138K), or SHP-1 with both SH2 domains inactivated (GST-SHP-1 R32K,R138K) was subjected to the same analysis as described for A. The beads were washed, and the bound tyrosine-phosphorylated proteins were visualized by SDS-PAGE and immunoblotting. All lanes are from the same blot with identical ECL exposure, but were rearranged for better clarity. Right panel, to analyze the SHP-1-associated tyrosine-phosphorylated proteins for glycosylation, beads with bound proteins were incubated in the absence or presence of endoglycosidases F/N (Endo F/N). The products were visualized by SDS-PAGE and immunoblotting using anti-phosphotyrosine antibodies. C: a GST-SHP-1 pull-down assay from lysates of EGF-stimulated A431 cells was performed as described for A. The anti-phosphotyrosine blot (upper panel) was stripped and reprobed with a monoclonal anti-p120ctn antibody (lower panel).

To identify pp100, we tested the possibility that pp100 may represent Gab1, c-Cbl, or Vav. Gab1 has been described to become tyrosine-phosphorylated in response to EGF and to bind SHP-2 effectively (60). Also, c-Cbl is tyrosine-phosphorylated in response to EGF, and this protein may serve as a docking partner for multiple SH2 domain proteins (61). Vav1 has been shown to functionally interact with SHP-1 in hematopoietic cells (62). Specific antibodies against Gab1, c-Cbl, Vav1, and Vav2 failed, however, to react with pp100 (data not shown).

We also considered the possibility that pp100 is a member of the recently described SIRP/SHP substrate family of docking proteins. A SIRP species of 85-90 kDa has been described in A431 cells (22). The SIRP species in A431 cells are heavily glycosylated membrane proteins that undergo a strong reduction in molecular mass upon deglycosylation with endoglycosidases (22). Treatment with endoglycosidases F/N of proteins bound to GST-SHP-1 did not result in a strong size change of the 100-kDa phosphoprotein band (Fig. 1B, right panel), in contrast to what would be expected for A431 cell-derived SIRP. The EGFR band was, however, shifted to lower molecular mass in this experiment, indicating that the endoglycosidase F/N treatment was effective. Furthermore, SIRP proteins in A431 cells are effectively bound to wheat germ agglutinin (22), whereas pp100 failed to bind to wheat germ agglutinin. Taken together, it seems unlikely that pp100 represents a member of the SIRP family.

Identification of pp100 as p120^ctn Isoform 3A-- Since pp100 seemed not to be identical to any of a number of candidate proteins, we aimed at direct microidentification of pp100 by chemical methods. Although pp100 could be visualized on silver- and Coomassie Blue-stained two-dimensional electrophoresis gels with an apparent isoelectric point of ~7 (data not shown), too little material could be recovered directly from these gels to allow unequivocal identification. Therefore, pp100 was partially purified using a two-step chromatography approach, and purification was monitored by pull-down assays with GST-SHP-1. In brief, A431 cells were stimulated with EGF and lysed, and tyrosine-phosphorylated proteins were enriched using anti-phosphotyrosine affinity chromatography. The phosphoproteins were further resolved by anion-exchange chromatography. The positive fractions were concentrated and loaded onto a one-dimensional SDS-polyacrylamide gel. The gel was stained with Coomassie Blue; and a band, tentatively assigned as pp100 on the basis of its size and enrichment properties, was excised. An in-gel digest was performed using trypsin, and the resulting peptide mixture was first analyzed using MALDI-TOF mass spectrometry. Sixty-seven peptide masses were determined and used to search the data base. The masses and parameters for the data base search are shown in Table I. With the peptide mass fingerprint data alone, an unequivocal identification of the eluted protein was possible. According to these data, the purified protein represented an isoform of human p120^ctn, a member of the Armadillo superfamily. This is supported by finding 31 peptide masses within a narrow error in the p120^ctn sequence, covering 42% of its sequence. A mass of 1051.58 may represent the p120^ctn-derived peptide with the sequence VVKAASGALR in its phosphorylated form. Phosphorylation of p120^ctn on serine residues has been reported previously (63). The remaining peptide masses were used to search the data base, but no significant match was observed.

We also resolved the peptide mixture from the in-gel digest by reversed-phase liquid chromatography and sequenced three peptides. The peptide sequences found (NLSYQVHR, EIPQAER, and GTTPLMQK) could be positioned in the human p120^ctn sequence, supporting the results obtained by mass spectrometry. The three peptide sequences were confirmed by MALDI-TOF of the isolated fractions.

We went on to verify the identity of pp100 to p120^ctn. An antibody raised against authentic p120^ctn recognized a 100-kDa protein in GST-SHP-1 pull-down assays (but not in GST mock precipitations), which exactly comigrated with pp100 (Fig. 1C). Furthermore, resolving immunoprecipitated p120^ctn from EGF-stimulated A431 cells using two-dimensional electrophoresis gels and immunoblotting with anti-phosphotyrosine or anti-p120^ctn antibodies yielded identical patterns as obtained with pp100 enriched by GST-SHP-1 pull-down assays (data not shown). Final identification of a p120^ctn isoform as the SHP-1-interacting pp100 was obtained by coexpression of authentic p120^ctn and SHP-1 and reconstitution of the molecular complex between both proteins (see below).

The expression of p120^ctn is regulated by alternative splicing, and at least 32 isoforms are theoretically possible (57). The antibody used recognizes all these isoforms of p120^ctn. RT-PCR with p120^ctn isoform-specific primers revealed that A431 cells express only the p120^ctn isoforms of type 3 containing sequences corresponding to exon A or B or both (Fig. 2A), i.e. 3A, 3AB, and/or 3B, with the isoform(s) containing exon B expressed to apparently lower levels. One of the sequenced pp100-derived peptides yielded the sequence GTTPLMQK, which does not occur in p120^ctn isoforms arising from expression of the alternative exon B. Also, isoform 3A migrated with identical mobility as pp100, whereas isoforms 3AB and 3B migrated with lower mobility than pp100 on SDS gels (Fig. 2B). Little immunoreactivity could be seen in lysates of EGF-stimulated A431 cells in the position of p120^ctn isoform 3AB or 3B. With the same type of lysates, antibodies specifically recognizing p120^ctn isoforms 1 and 2 yielded no signals, in agreement with the RT-PCR data indicating an absence of these isoforms. Antibodies specifically recognizing exon A-containing isoforms and antibodies specifically recognizing exon B-containing isoforms visualized strong or only weak bands, respectively, of ~100 kDa in corresponding immunoblots (data not shown), providing further evidence for a prevalence of p120^ctn isoform 3A expression in A431 cells. pp100 has an isoelectric point of ~7. This is close to the predicted isoelectric point of 6.7 for p120^ctn isoform 3A, whereas p120^ctn isoforms 3AB and 3B have predicted isoelectric points of 5.9. Taken together, it can be concluded that the SHP-1-interacting phosphoprotein pp100 from A431 cells represents p120^ctn isoform 3A. Our data do not, however, exclude that SHP-1 can bind, in addition, to other p120^ctn isoforms such as 3AB and 3B, which are expressed at comparatively low levels in A431 cells. Indeed, binding of SHP-1 to other p120^ctn isoforms could be shown in additional experiments (see below).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Analysis of p120ctn isoform expression in A431 cells. A, subconfluent cultures of A431 cells were starved and stimulated or not (as indicated) with EGF. Then, total RNA was isolated and subjected to RT-PCR analysis with primers indicating the presence or absence of alternatively used sequences in the expressed p120ctn isoforms, as shown. The patterns reveal expression of only type 3 isoforms and the absence of isoforms harboring sequences of exon C, but the presence of exon A and, to a lesser extent, exon B. B, lysates of EGF-stimulated 293 cells overexpressing EGFR and p120ctn isoform 3A, 3AB, or 3B (as indicated) and a lysate of EGF-stimulated A431 cells were analyzed by SDS-PAGE and immunoblotting with an antibody recognizing all p120ctn isoforms equally well. The main p120ctn isoform in A431 cells (pp100) comigrated with p120ctn isoform 3A. bp, base pairs.

Characterization of the SHP-1-p120^ctn Interaction-- We first analyzed whether the interaction between SHP-1 and p120^ctn was direct. To test this, p120^ctn was immunoprecipitated from lysates of EGF-stimulated A431 cells by anti-phosphotyrosine antibodies. These immunoprecipitates were denatured by boiling in the presence of 1% SDS. Under these conditions, any possible adaptor protein should be inactivated; however, the phosphotyrosine motifs in phosphorylated p120^ctn should retain at least part of their binding affinity for the SH2 domains of SHP-1. The denatured immunoprecipitates were diluted with lysis buffer to reduce the SDS concentration to 0.05% and subjected to a binding reaction with GST fusion proteins of SHP-1 (GST-SHP-1 WT), catalytically inactive SHP-1 (GST-SHP-1 CS), and the isolated tandem SH2 domains of SHP-1 (GST-SH2) or GST as a control. As demonstrated in Fig. 3, p120^ctn could be recovered on GST-SH2 and GST-SHP-1 WT to nearly the same extent, whereas no association with GST alone could be observed. This indicates that SHP-1 is capable of a direct interaction with phosphorylated p120^ctn via its SH2 domains. The use of catalytically inactive SHP-1 (GST-SHP-1 CS) dramatically increased the amount of coprecipitating p120^ctn. An explanation for this observation could be the efficient dephosphorylation of p120^ctn by SHP-1 during the binding reaction and therefore a removal of the interaction sites for the SH2 domains of SHP-1. Moreover, CS mutants of PTPs are known to trap substrates. The strong increase in binding of the SHP-1 CS mutant to p120^ctn compared with the GST-SH2 domains points to a second binding mechanism in addition to the SH2 domain-phosphotyrosine interaction, most likely an enzyme-substrate complex formation.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   SHP-1 binds directly to p120ctn. A431 cells were treated with EGF and subsequently lysed. Immunoprecipitation was carried out using anti-phosphotyrosine antibodies. To disrupt preexisting protein complexes, the immunoprecipitates were denatured in the presence of SDS. Subsequently, the denatured protein solution was diluted with a Triton X-100-containing buffer to a final SDS concentration of 0.05%. As indicated, the partially renatured proteins were incubated with either unfused GST or fusion protein GST-SHP-1 WT, GST-SHP-1 CS (catalytically inactive mutant), or GST-SH2 (isolated tandem SH2 domains), all immobilized on glutathione-Sepharose. The beads were washed, and the associated proteins were analyzed by SDS-PAGE and immunoblotting using anti-p120ctn antibodies (A). Equal levels of p120ctn in the denatured lysates could be revealed by SDS-PAGE and immunoblotting (B).

Differential Binding of SHP-1 to p120^ctn Isoforms-- As mentioned above, the expression of p120^ctn is regulated by alternative splicing, and this leads to a great variety of isoforms expressed in different cell lines. The major p120^ctn isoform interacting with SHP-1 in A431 cells was identified as p120^ctn isoform 3A. We wanted to further confirm the identity of pp100 by reconstituting the interaction using recombinant p120^ctn. Also, it was interesting to evaluate to what extent there might be a selectivity of certain p120^ctn isoforms for SHP-1 binding. Therefore, 11 different isoforms of p120^ctn, including p120^ctn isoform 3A, were tested for their ability to associate with SHP-1. The isoforms employed differed in the usage of the four translation start codons in the p120^ctn gene (designations 1-4) and/or in the presence or absence of the alternatively used exons A, B, and C. Isoform 3N contains no amino acid residues encoded by these alternative exons. The cDNAs of these isoforms were cotransfected with an expression plasmid for EGFR into 293 cells, after which the cells were stimulated with EGF for 5 min and lysed. The lysates were used in GST pull-down assays using GST-SHP-1 immobilized on glutathione-Sepharose. Total expression levels of the isoforms were comparable (Fig. 4B). The results of the pull-down assays presented in Fig. 4A indicate a different binding behavior of the various p120^ctn isoforms for SHP-1. Isoform 3A exhibited a very strong binding, further verifying its identity to pp100, whereas isoform 3AB bound clearly weaker. The lower molecular mass isoform 4A bound much more weakly to SHP-1 than isoforms 1A, 2A, and 3A, indicating that the N-terminal amino acids missing in isoform 4A are important for the association of p120^ctn with SHP-1. Interestingly, the presence of the small exon C (6 amino acids: DEWFSR) led to a strong reduction of association with SHP-1 in comparison with the association of the appropriate isoform without exon C (for instance, 3N versus 3C or 3A versus 3AC). One obvious explanation for the differential binding capacity of the different isoforms for SHP-1 could be a different efficiency of phosphorylation by the coexpressed and stimulated EGFR. Lysate aliquots were therefore used to monitor the phosphorylation of the various isoforms by immunoblotting. As shown in Fig. 4C, the various p120^ctn isoforms indeed became phosphorylated by EGFR with different efficiencies. Isoform 3A was a very good substrate for EGFR, which correlates with results from A431 cells, where this isoform (pp100) was found to be a major tyrosine-phosphorylated product after EGF stimulation. All isoforms containing the alternative exon C were phosphorylated to a much lesser extent compared with the corresponding p120^ctn isoforms without exon C (for instance, 3A versus 3AC). Very little if any phosphorylation was detectable in isoform 4A (Fig. 4C). Thus, weak binding of SHP-1 to isoforms 4A, 3AC, 3ABC, 3C, and 3BC is paralleled by poor tyrosine phosphorylation of these isoforms. In contrast, isoforms 3AB and 3B are effectively phosphorylated, but still exhibit comparatively little SHP-1 binding. Taken together, various p120^ctn isoforms have a differential capacity to bind SHP-1, which in part, but not entirely, correlates with their phosphorylation by coexpressed EGFR.

View larger version (48K): [in this window] [in a new window]   Fig. 4.   Various p120ctn isoforms exhibit differential binding to SHP-1. 293 cells were transfected with expression plasmids of different p120ctn isoforms (as indicated) and EGFR. After EGF stimulation, the cells were lysed, and lysates were applied to a GST pull-down assay employing immobilized GST-SHP-1. Bound proteins were visualized by SDS-PAGE and immunoblotting using anti-p120ctn antibodies (A). Similar expression levels for the various p120ctn isoforms were revealed by SDS-PAGE and immunoblotting (B). Lysate aliquots were loaded onto SDS-polyacrylamide gels, and the tyrosine phosphorylation of the different p120ctn isoforms was analyzed by SDS-PAGE and immunoblotting (C). Note that the order of some lanes in C differs from that in A and B. anti-PY, anti-phosphotyrosine.

p120^ctn has originally been described as a good substrate of the cytosolic tyrosine kinase Src (44). Since Src is endogenously expressed in A431 cells and becomes activated upon EGFR activation (64), it could possibly contribute to the EGF-mediated phosphorylation of p120^ctn. To explore this possibility, we performed inhibitor studies using compounds specifically inhibiting either the EGFR or Src kinase activity (Fig. 5). A431 cells were preincubated for 2 h with either the specific Src family kinase inhibitor PP1 or the specific EGFR kinase inhibitor AG1478 or with Me₂SO as the negative control. The cells were stimulated with EGF for 5 min or left unstimulated (as indicated) and finally lysed. The overall tyrosine phosphorylation was analyzed by running lysate aliquots on SDS-polyacrylamide gels and subsequent immunoblotting (Fig. 5, right panel). The tyrosine phosphorylation of p120^ctn was monitored after immunoprecipitation using anti-p120^ctn antibodies, followed by immunoblotting with anti-phosphotyrosine antibodies (Fig. 5, left panel). p120^ctn was found not to be phosphorylated on tyrosine residues in unstimulated cells. Preincubation of the cells with PP1 led to a partial reduction of the EGF-mediated p120^ctn phosphorylation to ~50% of the control (cells treated with Me₂SO), but not to a total loss of the phosphorylation. The EGF-induced overall tyrosine phosphorylation was also somewhat reduced after PP1 treatment. In contrast, inhibition of the EGFR kinase resulted in a complete abrogation of the EGF-mediated p120^ctn phosphorylation as well as the overall tyrosine phosphorylation. Therefore, Src either may be directly involved in the EGF-induced phosphorylation of p120^ctn subsequent to EGFR activation or cooperates with EGFR in a different way by influencing the ability of EGFR to phosphorylate p120^ctn.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   EGF-stimulated tyrosine phosphorylation of p120ctn is partially mediated by PP1-susceptible kinase(s). A431 cells at 70% confluence were treated with PP1 (1 µM), AG1478 (0.3 µM), or dimethyl sulfoxide (DMSO) for 2 h. The cells were subsequently stimulated with EGF or vehicle (as indicated) and lysed. p120ctn was immunoprecipitated (IP), and its tyrosine phosphorylation was analyzed by SDS-PAGE and immunoblotting (left panel). The overall tyrosine phosphorylation was visualized by running lysate aliquots on SDS gels and immunoblotting (right panel). anti-PY, anti-phosphotyrosine.

Association with SHP-1 and Dephosphorylation of p120^ctn by SHP-1 in A431 Cells-- We next asked whether complex formation between p120^ctn and SHP-1 can be shown in intact cells. To test this, we employed stably transfected A431 cells that express SHP-1 under the control of a tetracycline-dependent promoter in addition to comparatively low levels of endogenous SHP-1. In these cells, the presence of ATc represses the expression of SHP-1, and withdrawal of ATc leads to an induction of SHP-1 expression ("tet-off" system) (54). Such A431 cells, grown in the absence of ATc and thus expressing SHP-1, were stimulated for different time periods with EGF. SHP-1 was immunoprecipitated, and any associated p120^ctn was analyzed by SDS-PAGE and immunoblotting. The results presented in Fig. 6 indicate that p120^ctn formed a complex with SHP-1 but only when the cells were stimulated with EGF. The complex formation was rapid and occurred already after a 30-s stimulation with EGF. Furthermore, the association was of transient nature, peaking at ~5 min and declining after 10 min. Similar experiments were performed with untransfected A431 cells expressing low levels of endogenous SHP-1 and with ZR75-1 mammary carcinoma cells expressing high amounts of endogenous SHP-1. We failed, however, to visualize complex formation in these cell lines (data not shown). It may be relevant that p120^ctn is only poorly phosphorylated after EGF stimulation of ZR75-1 cells.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   p120ctn becomes physically associated with SHP-1 in A431 cells upon EGF stimulation. A431 cells stably expressing SHP-1 were stimulated with EGF for the indicated time periods. The cells were lysed, and SHP-1 was immunoprecipitated. Associated p120ctn was visualized by SDS-PAGE and immunoblotting using anti-p120ctn antibodies (A). Similar expression levels for p120ctn were revealed by running lysate aliquots on SDS-polyacrylamide gels and subsequent immunoblotting (B). Mab, monoclonal antibody.

The data on the interaction between p120^ctn and SHP-1 presented above suggested the possibility that p120^ctn might be an efficient substrate for SHP-1. To test this, SHP-1-expressing A431 cells (+SHP-1) and mock-transfected cells (SHP-1) were grown in the absence or presence of ATc and stimulated with EGF for different time periods or left unstimulated, as indicated in Fig. 7. After this, the cells were lysed; p120^ctn was immunoprecipitated; and the tyrosine phosphorylation of the protein was visualized by immunoblotting. Already after 1 min of EGF stimulation, p120^ctn became strongly tyrosine-phosphorylated. This phosphorylation was constant up to 10 min of EGF stimulation. In control cells, the presence or absence of ATc had no effects on the EGF-induced p120^ctn tyrosine phosphorylation. In cells transfected with the SHP-1 expression construct in the presence of ATc (suppressed SHP-1 expression), a somewhat lower phosphorylation of p120^ctn was observed as compared with the mock-transfected cells. This may be due to clonal variation or to some vector leakage. Indeed, even in the presence of ATc, a small amount of SHP-1 could be detected (Fig. 7C), which might be sufficient to partially dephosphorylate p120^ctn. More important, upon withdrawal of ATc, the clear induction of SHP-1 was accompanied by a clear reduction of the p120^ctn phosphorylation. These results suggest that in intact cells, p120^ctn is not only a binding partner, but also a substrate for SHP-1.

View larger version (58K): [in this window] [in a new window]   Fig. 7.   p120ctn is dephosphorylated by SHP-1 in A431 cells inducibly expressing SHP-1. A431 cells stably and inducibly expressing SHP-1 (+SHP-1) or mock-transfected cells (SHP-1) were treated for 5 days with 100 ng/ml ATc or vehicle to either suppress or induce SHP-1, respectively. The cells were treated with EGF for different time periods (as indicated), and lysates were prepared. p120ctn was immunoprecipitated, and its tyrosine phosphorylation was visualized by SDS-PAGE and immunoblotting using anti-phosphotyrosine (anti-PY) antibodies (A). Expression of p120ctn (B) and SHP-1 (C) was revealed by running lysate aliquots on SDS-polyacrylamide gels and immunoblotting.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Multiple targets for the protein-tyrosine phosphatase SHP-1 have been identified in hematopoietic cells (for reviews, see Refs. 2-5). In contrast, little is known about the function of SHP-1 in epithelial cells. In this study, we have identified p120^ctn as a binding partner and substrate for SHP-1 in A431 epidermoid carcinoma cells. Epithelial isoforms of p120^ctn interact with E-cadherin and are components of adherens junctions (30, 32). One could speculate that SHP-1 may have the capacity to regulate p120^ctn function and, in turn, cell-cell adhesion. Unfortunately, although p120^ctn is a prominent tyrosine kinase substrate and, in fact, one of the most abundantly phosphorylated proteins in A431 cells upon EGF stimulation, the role of its tyrosine phosphorylation is unclear. p120^ctn has been shown to modulate cadherin-mediated cell-cell adhesion negatively in certain cell types (50, 51), and phosphorylation could possibly modify this activity. We did, however, not observe any effects of SHP-1 expression on cell-cell adhesion of A431 cells under the assay conditions employed.² Phosphorylation of p120^ctn by Src has recently been reported to increase its binding to E-cadherin in vitro (65). One could therefore speculate that dephosphorylation of tyrosine-phosphorylated p120^ctn by SHP-1 would decrease its affinity for E-cadherin. Isoforms of p120^ctn lacking sequences corresponding to exon B have the capacity to localize to the nucleus and may regulate transcription of specific genes (52). Tyrosine phosphorylation of p120^ctn could potentially affect its cellular localization. The SHP-1 expression level had, however, little effect on cellular localization of p120^ctn in A431 cells as visualized by indirect immunofluorescence.² A better understanding of p120^ctn function in epithelial cells and of the role of p120^ctn tyrosine phosphorylation is required to more specifically explore the functional effects of its SHP-1 association and SHP-1-mediated dephosphorylation.

Pull-down assays with SDS-denatured p120^ctn and with various SHP-1 mutant proteins revealed that the association is at least partially direct and mediated to a greater extent by the N-terminal SH2 domain as compared with the C-terminal SH2 domain. Interestingly, the catalytically inactive C455S mutant of SHP-1 exhibited greatly elevated binding, suggesting that p120^ctn is a very efficient SHP-1 substrate even under the conditions of the pull-down assay. Two tyrosine residues in the sequence of p120^ctn partially resemble the consensus sequence for ligands of the N-terminal SH2 domain of SHP-1, i.e. hXY(P)XXh (where h = hydrophobic and X = any amino acid). Tyrosines 549 (RGY⁵⁴⁹ELL, numbering according to isoform 3A) and 561 (RIY⁵⁶¹ISL) in their phosphorylated forms are therefore candidates for SHP-1 interaction sites in p120^ctn. These putative binding sites for SHP-1 in p120^ctn are present in all p120^ctn isoforms. We did, however, observe a differential binding of SHP-1 to 11 tested p120^ctn isoforms. This differential binding can in part be attributed to differential phosphorylation of the various isoforms. Interestingly, p120^ctn sequences corresponding to exon C had apparently a negative influence on tyrosine phosphorylation of the respective p120^ctn isoforms. Also, isoform 4A, which lacks N-terminal sequences present in isoforms 3, was very poorly phosphorylated and consequently bound very little p120^ctn. Exon C sequences may negatively affect interaction of p120^ctn with the phosphorylating kinase(s). It is noteworthy that exon C is largely brain-specific (57). Alternatively, N-terminal sequences missing in isoform 4A may positively affect such interaction with a tyrosine kinase. The identity of the latter is not clear. Although the tyrosine phosphorylation of p120^ctn in A431 cells is strictly dependent on the activity of EGFR kinase, as indicated by total abolishment with the selective EGFR blocker AG1478, the Src family kinase inhibitor PP1 showed a partial inhibitory effect. This finding may be due to the fact that Src family kinases partially contribute to EGF-stimulated p120^ctn phosphorylation or may be due to only partial inhibition of an EGF-dependent p120^ctn-modifying kinase by PP1. For example, susceptibility to PP1 of the cytoplasmic tyrosine kinase FER, which has been shown to efficiently phosphorylate p120^ctn upon overexpression or in a receptor tyrosine kinase-dependent manner (46), is not known. Alternatively, Src family kinase inhibition may partially impair EGFR tyrosine kinase activity. Src kinase has been shown to contribute to full functionality of EGFR (66, 67). Interestingly, also the presence of p120^ctn sequences corresponding to exon B had a negative effect on SHP-1-p120^ctn association, although the overall phosphorylation of exon B-containing isoforms was comparable to that without exon B. The presence of exon B sequences seems therefore to impair SHP-1 binding independently of tyrosine phosphorylation.

In A431 cells, p120^ctn becomes effectively dephosphorylated upon induction of SHP-1 expression. Complex formation between SHP-1 and p120^ctn appears to be very transient, and SHP-1-mediated dephosphorylation is probably the cause of rapid complex decomposition. This may also be the reason why we were unable to visualize SHP-1-p120^ctn complexes in cells with endogenous SHP-1 levels. To demonstrate control of p120^ctn tyrosine phosphorylation by endogenous SHP-1 will require experiments to down-regulate endogenous SHP-1 levels.

The observed association of SHP-1 with p120^ctn in A431 cells and its dephosphorylation by SHP-1 provide strong evidence for the first demonstrated interaction of a PTP with this prominently tyrosine-phosphorylated member of the Armadillo superfamily (see "Note Added in Proof"). We propose that SHP-1 has the capacity to modulate p120^ctn function by dephosphorylation. This may not apply only to epithelial cells. As we have shown, SHP-1 has the capacity to interact with a variety of p120^ctn isoforms, in addition to isoform 3A. Various p120^ctn isoforms have, for example, been detected in a murine macrophage cell line (63), and murine macrophages are known to express relatively high levels of SHP-1. One could therefore speculate that SHP-1 can also modulate p120^ctn tyrosine phosphorylation levels in other cell types where both molecules are coexpressed.

	ACKNOWLEDGEMENTS

We are very grateful to Drs. A. Ullrich and R. Lammers for the generous provision of SHP-1 cDNA and also to Christer Wernstedt (Ludwig Institute Uppsala) for accurate amino acid sequence determination.

	Note Added in Proof

While this paper was under review, Zondag et al. (Zondag, G. C. M., Reynolds, A. B., and Moolenaar, W. H. (2000) J. Biol. Chem. 275, 11264-11269) reported association of p120^ctn with, and dephosphorylation by, the receptor-like protein-tyrosine phosphatase RPTPµ.

	FOOTNOTES

^* This work was supported by Grant Bo 1043/3-1 from the Deutsche Forschungsgemeinschaft (to F.-D. B. and J. G.-Z.) and by a grant from the Max-Planck Society (to F.-D. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Postdoctoral Fellow of the Fund for Scientific Research, Flanders.

^** Research Director of the Fund for Scientific Research, Flanders.

^§§ Present address: Centre for Molecular Medicine, University College London, 5 University Street, London WC1E 6JJ, UK.

^¶¶ To whom correspondence should be addressed. Fax: 49-3641-304462; E-mail: i5frbo@rz.uni-jena.de.

Published, JBC Papers in Press, June 1, 2000, DOI 10.1074/jbc.M001315200

² H. Keilhack, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PTP, protein-tyrosine phosphatase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GST, glutathione S-transferase; WT, wild-type; PAGE, polyacrylamide gel electrophoresis; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; RT-PCR, reverse transcription-polymerase chain reaction; ATc, anhydrotetracycline; SIRP, signal-regulating protein.

	REFERENCES

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